1) We have an ongoing project to develop improved green photoactivatable fluorescent proteins (PAFPs) for Photoactivated Localization Microscopy (PALM). Currently, PAFPs are available in essentially two colors, green and red. Red PAFPs have proven to be useful for PALM by providing low backgrounds and sufficient numbers of photons capable of <25 nm uncertainty in localization. Green PAFPs suffer from low photons and high backgrounds, which limit precise molecular localization. We are currently working on developing an improved green PAFP for PALM. Previous improvements included variants of PAGFP with low background, but at the expense of photoactivation contrast. Efforts are aimed at reengineering by site-directed mutagenesis high photoactivation contrast while maintaining the low background fluorescence have produced molecules with both low background and high contrast, but now at the expense of brightness. Yan Fu is working on this project and currently further characterizing candidate molecules and recover brightness in the mutants of interest. 2) A project related to PAFPs as well as conventional fluorescent proteins, whether current or under development, is the characterization of their blinking behaviors. Ideally, PAFPs would be turned on, fluoresce for a given period of time, photobleach, and never turn on again. However, common to almost all fluorophores, PAFPs sample dark states during which they do not produce photons. This is problematic since it is difficult to determine if a molecule has blinked or photobleached, and when it turns on once again it is difficult to determine if it is the same molecule or a different molecule. Simulations of blinking behavior indicate that these can dramatically affect molecule counting (density determinations) as well structural interpretations. We have devised assays and analyses to survey existing molecules in efforts to determine parameters that can correctly identify blinking molecules, combine their collected photons and correctly localize them only once. In addition, we are testing variants with mutations within and around the PAFP chromophores to determine which affect the blinking on times, blinking off times, and percentage of blinking molecules in a population. This project currently involves Yan Fu and Ernesto Casillas (NIH Academy trainee). Collaborators on the project include the lab of Jennifer Lippincott-Schwartz (NICHD). 3) We are collaborating with Moshe Levi (UCHSC) on a project to image with PALM the localization of sodium phosphate transporters, NaPi-2a and NaPi-2c, in the apical brush border membrane (BMM) of opossum kidney (OK) cells. These molecules play key roles in renal proximal tubule inorganic phosphate (Pi) reabsorption and help maintain Pi homeostasis. These transporters respond to increases and decreases in dietary Pi by changing their abundance in the BMM via trafficking to and from the BMM and an intracellular compartment. The transporters are known to interact with a number of PDZ containing proteins, such as NHERF-1 and NHERF-3, and to be associated with microdomains enriched in cholesterol and glycosphingolipids. Our project will require multi-color PALM experiments on NaPi molecules and their interacting partners located in the convoluted apical membrane of OK cells under several conditions affecting molecule localization, molecule diffusion, and molecule clustering. Thus, we will need to localize the molecules in 3 dimensions within > 1m long membrane microvilli structures. These will be challenging experiments from both imaging and biology standpoints, but the obstacles are straightforward and we anticipate overcoming them as the project matures. The PAFP tagged molecules have been tested by expression in COS 7 cells and now also in the more relevant OK cells. The localization of the tagged molecules is conforming to the normal localization of the NaPi proteins and we have collected numerous 2D and 3D PALM data sets on cells expressing single PAFP-tagged molecule. Experiments are currently underway to collect data on multiple combinations of PAFP-tagged proteins of interest. Plasmids have also been developed and tested thoroughly for expressing two molecules from the same promoter to better control relative expression levels and these are being used whenever appropriate. This project is being run by Kristin Rainey. 4) Our collaboration with Nihal Altan-Bonnet involves PALM imaging of plus strand RNA viral infection of cells. These viruses include picornaviruses, coronaviruses, and flaviviruses, which after infection of a cell dramatically remodel intracellular membranes into replication organelles on which RNA replication takes place. A number of intracellular proteins are hijacked during this process and recruited to the replication membranes. Recent studies from the Altan-Bonnet lab have identified an important role for phosphotidylinositol 4 phosphate (PI4P) lipids in regulating the RNA replication by recruiting the viral RNA polymerase. The development of these PI4P membrane microdomains requires recruitment of phophatidylinositol-4-kinase IIIb (PI4KIIIb), which is recruited by the small Ras-family GTPase Arf1. The activity of Arf1 and its guanine nucleotide exchange factor GBF1 are modulated by 3A, a viral tail-anchored membrane protein. Our interest in this project is to image at super-resolution levels (< 50nm) the localization of the numerous viral and host cell components on the replication organelles involved in replication at various times points post-infection. This project presents many of the same challenges as the NaPi project. This project is currently observing host cell and viral components in uninfected and coxsackievirus infected cells. Yan Fu is now running this project. 5) We collaborate with Anamaris Colberg-Poley on super-resolution imaging of human cytomegalovirus infected cells. Our interest is gaining insight into the transfer of pUL37x1 protein from mitochondria associated membranes to the outer mitochondria membrane. Data have been collected on single expressed proteins using uninfected cells expressing PAFP tagged versions of the pUL37x1. Ongoing work is shifting to 2-color imaging to compare the localizations of multiple proteins of interest. This project has been transferred to Ernesto Casillas. 6) We collaborate with Wei-Shau Hu (NCI) on a project to localize the centers of mass of several proteins and RNAs within noninfectious Human Immunodeficiency Virus (HIV) virus-like particles. Genes for several critical components, Env, Vif, Vpu, and Vpr, have large deletions making the particles noninfectious. The lab of Wei-Shau Hu has developed several variants of these particles containing multiple color conventional fluorescent protein tagged components. For several of these particles the fluorescence labels are fusion proteins (CFP, YFP, and mCherry) which target RNA stem loops that have been engineered into different regions of the genomic RNA. Since the particles (<100 nm diameter) are smaller than the resolution limit of conventional optics, the distribution of the different molecules appears as a diffraction-limited 500 nm spot. However, by two-dimensional Gaussian fitting of the fluorescence distributions, we can provide precise localization of the average localization of the molecules. This differs from PALM since all molecules are imaged at once, but for the initial steps in this project we wish to simply define the relative positions of the particle components. This project is under the guidance of Maria Ingaramo with assistance from Mary Sun. Maria has made significant steps in lowering our localization errors and we should soon have sufficient precision to adequately determine mean distances from the centers of mass for several labeled components.